Genomic Analysis After Serial Passages In Duck Embryo And Construction Of Infectious Clone Of A Novel Goose Parvovirus

Since 2014,a disease characterized by stunted development,atrophy of upper and lower beaks,tongue extension,swelling and downward bending of ducklings,commonly known as "short beak and dwarfism syndrome"(SBDS),has appeared in commercial duck flocks in Jiangsu,Shandong and Hebei regions of China,to China's poultry industry has caused huge economic losses.The pathogen of the disease is a novel goose parvovirus(NGPV),also known as duck goose parvovirus,novel duck parvovirus,etc.At present,the key genes that determine the virulence of NGPV,and the causes of cross-species transmission of NGPV are still unclear.To obtain an attenuated NGPV strain,in this study,the NGPV SD strain was passed through successive generations of duck embryos to the F50 generation.In this study,the F5 allantoic fluid and F50 allantoic fluid were respectively infected with 3-day-old Cherry Valley ducks for pathogenicity test.The body weight,beak length and detoxification of the infected ducks were measured.The body weight of the F5 infection group and the control group was very significant at 14 dpi,the beak length was already different at 7 dpi,and it was very significant at 14 dpi,and all ducks have virus shedding at 3 dpi;the body weight of the F50 infection group and the control group show a difference at 14 dpi,and it was extremely significant at 21 dpi,the beak length was already different at 7 dpi,and it was very significant at 14 dpi,and all ducks have virus shedding at 5 dpi.In summary,the F50 generation is significantly less toxic than the F5 generation.In order to investigate the key virulence genes of NGPV,in this study,the whole genome of SD-F10?SD-F20?SD-F30?SD-F40 and SD-F50 was also sequenced,and the sequence alignment analysis revealed that SD-F50 could cover the base changes of all previous generations,and the base sequences of SD-F50 were compared with SD strain,and it was found that G26A,C61T,G320A,C355T mutations occurred in SD-F50 compared with SD strain at the ITR site,and the In the NS gene,SD-F50 had A1184C mutation compared with the SD strain;in the VP gene,SD-F50 had A3865G,C4521A and C2622T mutations compared with the SD strain.Comparing the amino acid sequences of the NS and VP proteins of the SD strain,in the NS gene,the mutation in SD-F50 compared to the SD wild strain was N225T,and in the VP gene,the mutations in SD-F50 compared to the SD wild strain were T465A and N703K.These mutations may affect the virulence of NGPV.To further investigate the relationship between these mutant loci and pathogenicity,a full-length infectious cloning plasmid of the SD strain was constructed in this study.A genetic molecular marker was introduced at position 3807 of the genome by synonymous mutation to eliminate the Nco I enzyme cut site as a distinguishing site from the wild virus strain.By simultaneously transfecting the duck embryo urinary bladder cavity and yolk sac,and identifying the virus,we finally determined the successful rescue of the virus,and named the rescue virus as rSD.In this study,whole genome sequence analysis of the passaged strain of NGPV SD strain revealed that the genome sequence of SD strain underwent some adaptive mutations with the passing of duck embryos,and these mutations might be related to the virulence genes of NGPV and the adaptation of NGPV to ducks,which could provide a reference basis for the research about the key genes of NGPV virulence.Meanwhile,the successful construction of infectious clones of NGPV SD strains may also provide technical support for subsequent studies on NGPV virulence factors and cross-species transmission.